Species-specific functional interactions of DNA polymerase alpha-primase with simian virus 40 (SV40) T antigen require SV40 origin DNA.

Physical and functional interactions of simian virus 40 (SV40) and polyomavirus large-T antigens with DNA polymerase alpha-primase were analyzed to elucidate the molecular basis for the species specificity of polymerase alpha-primase in viral DNA replication. SV40 T antigen associated more efficiently with polymerase alpha-primase in crude human extracts than in mouse extracts, while polyomavirus T antigen interacted preferentially with polymerase alpha-primase in mouse extracts. The apparent species specificity of complex formation was not observed when purified polymerase alpha-primases were substituted for the crude extracts. Several functional interactions between T antigen and purified polymerase alpha-primase, including stimulation of primer synthesis and primer elongation on M13 DNA in the presence or absence of the single-stranded DNA binding protein RP-A, also proved to be independent of the species from which polymerase alpha-primase had been purified. However, the human DNA polymerase alpha-primase was specifically required for primosome assembly and primer synthesis on SV40 origin DNA in the presence of T antigen and RP-A.     

Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen.

The purified human single-stranded DNA binding protein, replication protein A (RP-A), forms specific complexes with purified SV40 large T antigen and with purified DNA polymerase alpha-primase, as shown by ELISA and a modified immunoblotting technique. RP-A associated efficiently with the isolated primase, as well as with intact polymerase alpha-primase. The 70 kDa subunit of RP-A was sufficient for association with polymerase alpha-primase. Purified SV40 large T antigen bound to intact RP-A and to polymerase-primase, but not to any of the separated subunits of RP-A or to the isolated primase. These results suggest that the specific protein-protein interactions between RP-A, polymerase-primase and T antigen may play a role in the initiating of SV40 DNA replication.     

Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro.

DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster. This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication. The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells. This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA. By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A. In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase alpha-primase. These results collectively indicate that many of the specific functional interactions among T antigen, polymerase alpha-primase, and RP-A are conserved from primates to Drosophila species. Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.     

Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase.

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.     

Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex.

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.     

Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase.

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.     

Species specificity of simian virus 40 DNA replication in vitro requires multiple functions of human DNA polymerase alpha

Human cell extracts support the replication of SV40 DNA, whereas mouse cell extracts do not. Species specificity is determined at the level of initiation of DNA replication, and it was previously found that this requires the large subunit, p180, of DNA polymerase alpha-primase to be of human origin. Furthermore, a functional interaction between SV40 large T antigen (TAg) and p180 is essential for viral DNA replication. In this study we determined that the N-terminal regions of human p180, which contain the TAg-binding sites, can be replaced with those of murine origin without losing the ability to support SV40 DNA replication in vitro. The same substitutions do not prevent SV40 TAg from stimulating the activity of DNA polymerase alpha-primase on single-stranded DNA in the presence of replication protein A. Furthermore, biophysical studies show that the interactions of human and murine DNA polymerase alpha-primase with SV40 TAg are of a similar magnitude. These studies strongly suggest that requirement of SV40 DNA replication for human DNA polymerase alpha depends neither on the TAg-binding site being of human origin nor on the strength of the binary interaction between SV40 TAg and DNA polymerase alpha-primase but rather on sequences in the C-terminal region of human p180.     

The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.     

Stimulation of mouse DNA primase-catalyzed oligoribonucleotide synthesis by mouse DNA helicase B.

Many prokaryotic and viral DNA helicases involved in DNA replication stimulate their cognate DNA primase activity. To assess the stimulation of DNA primase activity by mammalian DNA helicases, we analyzed the synthesis of oligoribonucleotides by mouse DNA polymerase alpha-primase complex on single-stranded circular M13 DNA in the presence of mouse DNA helicase B. DNA helicase B was purified by sequential chromatography through eight columns. When the purified DNA helicase B was applied to a Mono Q column, the stimulatory activity for DNA primase-catalyzed oligoribonucleotide synthesis and DNA helicase and DNA-dependent ATPase activities of DNA helicase B were co-eluted from the column. The synthesis of oligoribonucleotides 5-10 nt in length was markedly stimulated by DNA helicase B. The synthesis of longer species of oligoribonucleotides, which were synthesized at a low level in the absence of DNA helicase B, was inhibited by DNA helicase B. The stimulatory effect of DNA helicase B was marked at low template concentrations and little or no effect was observed at high concentrations. The mouse single-stranded DNA binding protein, replication protein A (RP-A), inhibited the primase activity of the DNA polymerase alpha-primase complex and DNA helicase B partially reversed the inhibition caused by RP-A.     

The mouse DNA polymerase alpha-primase subunit p48 mediates species-specific replication of polyomavirus DNA in vitro.

Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human cell extracts do not. However, the addition of purified mouse DNA polymerase alpha-primase to human cell extracts renders them permissive for Py DNA replication, suggesting that mouse polymerase alpha-primase determines the species specificity of Py DNA replication. We set out to identify the subunit of mouse polymerase alpha-primase that mediates this species specificity. To this end, we cloned and expressed cDNAs encoding all four subunits of mouse and human polymerase alpha-primase. Purified recombinant mouse polymerase alpha-primase and a hybrid DNA polymerase alpha-primase complex composed of human subunits p180 and p68 and mouse subunits p58 and p48 supported Py DNA replication in human cell extracts depleted of polymerase alpha-primase, suggesting that the primase heterodimer or one of its subunits controls host specificity. To determine whether both mouse primase subunits were required, recombinant hybrid polymerase alpha-primases containing only one mouse primase subunit, p48 or p58, together with three human subunits, were assayed for Py replication activity. Only the hybrid containing mouse p48 efficiently replicated Py DNA in depleted human cell extracts. Moreover, in a purified initiation assay containing Py T antigen, replication protein A (RP-A) and topoisomerase I, only the hybrid polymerase alpha-primase containing the mouse p48 subunit initiated primer synthesis on Py origin DNA. Together, these results indicate that the p48 subunit is primarily responsible for the species specificity of Py DNA replication in vitro. Specific physical association of Py T antigen with purified recombinant DNA polymerase alpha-primase, mouse DNA primase heterodimer, and mouse p48 suggested that direct interactions between Py T antigen and primase could play a role in species-specific initiation of Py replication.     

Primer-DNA formation during simian virus 40 DNA replication in vitro.

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.     

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) alpha-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis. Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated. Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin. These studies provide additional evidence that the Pol alpha-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated.     

Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase alpha-primase.

Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase alpha-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA replication of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase alpha-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex.     

Purification and functional characterization of bovine RP-A in an<Emphasis Type="Italic">in vitro</Emphasis> SV40 DNA replication system

The single-stranded DNA binding protein RP-A is required in SV40 DNAin vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replicationin vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase α-primase complex.     

Identification of cellular proteins required for simian virus 40 DNA replication

Study of the proteins involved in DNA replication of a model system such as SV40 is a first step in understanding eukaryotic chromosomal replication. Using a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication, we conducted a series of systematic fractionation-reconstitution experiments for the purpose of identifying and characterizing the cellular proteins involved in SV40 DNA replication. In addition to the one viral-encoded replication protein, T antigen, we have identified and begun to characterize at least six cellular components from a HeLa cytoplasmic extract that are absolutely required for SV40 DNA replication in vitro. These include: (i) two partially purified fractions, CF IC and CF IIA, and (ii) four proteins that have been purified to near homogeneity, replication protein-A, proliferating cell nuclear antigen, DNA polymerase alpha-primase complex, and topoisomerase (I and II). Replication protein-A is a multi-subunit protein that has single-stranded DNA binding activity and is required for a T antigen-dependent, origin-dependent unwinding reaction which may be an important early step in initiation of replication. Fraction CF IC can stimulate this unwinding reaction, suggesting that it also may function during initiation. Proliferating cell nuclear antigen, DNA polymerase alpha-primase, and CF IIA all appear to be involved in elongation of nascent chains.     

Synthesis of DNA by DNA polymerase epsilon in vitro.

The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.     

Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.

Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases alpha and delta. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase alpha activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases alpha and delta. The primer recognition complex, RF-C.PCNA.ATP, stimulated pol delta activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase alpha. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase alpha dependent synthesis of lagging strands and DNA polymerase delta/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.     

An N-terminal deletion mutant of simian virus 40 (SV40) large T antigen oligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNA polymerase alpha and replicate SV40 DNA in vitro.

A peptide encompassing the N-terminal 82 amino acids of simian virus 40 (SV40) large T antigen was previously shown to bind to the large subunit of DNA polymerase alpha-primase (I. Dornreiter, A. Höss, A. K. Arthur, and E. Fanning, EMBO J. 9:3329-3336, 1990). We report here that a mutant T antigen, T83-708, lacking residues 2 to 82 retained the ability to bind to DNA polymerase alpha-primase, implying that it carries a second binding site for DNA polymerase alpha-primase. The mutant protein also retained ATPase, helicase, and SV40 origin DNA-binding activity. However, its SV40 DNA replication activity in vitro was reduced compared with that of wild-type protein. The reduction in replication activity was accompanied by a lower DNA-binding affinity to SV40 origin sequences and aberrant oligomerization on viral origin DNA. Thus, the first 82 residues of SV40 T antigen are not strictly required for its interaction with DNA polymerase alpha-primase or for DNA replication function but may play a role in correct hexamer assembly and efficient DNA binding at the origin.     

Mutational analysis of simian virus 40 T-antigen primosome activities in viral DNA replication

The recruitment of DNA polymerase alpha-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.     

Complete enzymatic synthesis of DNA containing the SV40 origin of replication

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.